In this supplemental project, we seek to capitalize on a confluence of exciting new data, innovative technology, and the unique expertise of three new collaborators. Data from our parent project have identified an obscure sphingomyelinase isoform -- neutral sphingomyelinase-3 (nSMase-3) -- as a potential mediator of diaphragm weakness stimulated by TNF or severe heart failure. We propose to test this thesis by a multidisciplinary strategy. The experimental approach includes cell culture systems, a newly-created transgenic mouse line, and diaphragm biopsies from humans with heart failure. The project is made possible by a powerful team of three new collaborators who are experts in molecular biology, transplantation surgery, and human genetics. The goal of our project is to evaluate nSMase-3 as a potential mediator of diaphragm weakness in heart failure and a target for future therapeutic development. We have two specific aims: Aim 1. To evaluate nSMase-3 as a mediator of TNF-induced oxidant activity and weakness. Experiment 1.1 will test the hypothesis that nSMase-3 is constitutively expressed by murine skeletal muscle and is upregulated by TNF. Experiment 1.2 will test the hypothesis that muscle-specific nSMase-3 deficiency will abolish TNF effects on diaphragm oxidant activity and specific force. Aim 2. To test for associations between nSMase-3 and cardiovascular disease in humans. Experiment 2.1 will test the hypothesis that nSMase-3 mRNA, nSMase-3 protein, and SMase activity are elevated in diaphragm and pectoralis muscles of patients undergoing surgery for heart failure. Experiment 2.2 will test the hypothesis that single nucleotide polymorphisms (SNPs) in the Smpd4 gene that codes for nSMase-3 will modulate expression or splicing of the gene in human tissue. PUBLIC HEALTH RELEVANCE: Weakness and fatigue of the respiratory muscles cause exercise intolerance, breathlessness, and respiratory failure in patients with chronic heart and lung diseases. This supplemental project tests a novel enzyme, neutral sphingomyelinase-3, as a possible cause of respiratory muscle dysfunction in chronic disease and a potential target for future therapies.